PSMC3Prevents Human Argonaute2from Proteasomal Degradation

Objective:RNA interference is a type of gene silencing mediated by double-stranded RNA (dsRNA). Either endogenous or exogenous small RNAs can cooperate with Argonaute and associated factors into RNA-induced silencing complex (RISC) by combining with their target mRNAs. Finally, RISC can result in cleavage, degradation or translation repression of target mRNAs. In previous study, we employed the CytoTrap two-hybrid system to screen a human lung cDNA library using human Ago2PIWI domain as bait. The specific interaction of PSMC3with Ago2was examined by coimmunoprecipitation. Here we further define the interaction domains between PSMC3and Ago2, and explore the effect of PSMC3on Ago2expression and the consequences of Ago2function.Methods:Yeast two-hybrid and coimmunoprecipitation were used to map the interaction of PSMC3and Ago2. After PSMC3was ectopic expressed or depleted by specific siRNA, we checked endogenous Ago2protein level by western blot. In addition, cycloheximide was used to investigate Ago2protein tuneover, and MG132was apply to analyse the role of26S proteasomal degradation in Ago2protein stability. With the help of EGFP-based positive readout reporter system, we study the effect of PSMC3knockdown or overexpression on siRISC and miRISC activities.Result:1. The Coiled-Coil region in PSMC3N terminal is identified to be involved in Ago2-PSMC3interaction.2. Overexpression of PSMC3enhanced Ago2protein level, and depletion of PSMC3by siRNAs reduced Ago2protein level.3. Depletion of PSMC3could increase ubiquitylation of Ago2and result in Ago2degradation. 4. The reduced Ago2level by PSMC3kockdown was overrided by the proteasome inhibitor MG132.5. EGFP reporter system showed siRNA and miRNA mediated mRNA cleavage activity was suppressed by knocking down PSMC3in HeLa stable cell lines.Conclusion:The Coiled-Coil region of PSMC3is found to be necessary for the physical interaction with Ago2, and PSMC3-Ago2interaction is involved in regulating Ago2stability. Depletion of PSMC3in HeLa cells resulted in ubiquitylation of Ago2. Proteasome degradation is responsible for the deregulation of Ago2protein level and repression of RISC activity. As Ago2is a crucial component of RISC, identification of a novel Ago2partner will help us to understand the key factors in RNAi Pathway and elucidate its complex molecular mechanism.